PROJECT SUMMARY Pediatric cancers can be considered developmental disorders in which oncogenic drivers hijack normal developmental programs to promote tumorigenesis. The scaffolding protein menin is essential for normal development and, in human cancer, can function as a tumor suppressor or an oncogene, depending on context. The diverse functions of menin are linked to its role in regulation of gene transcription via its interaction with MLL histone methyltransferases, as well as with other context-dependent binding partners. In MLL-rearranged leukemia (MLLr), protein:protein interactions between menin and MLL-fusion proteins drive epigenetic activation of oncogenic transcription programs. A critical dependence of MLLr leukemia on these interactions represents a unique therapeutic vulnerability and small molecule inhibitors of the menin:MLL interaction are being developed for leukemia-directed therapy. Ewing sarcomas are mesenchymal tumors of presumed stem cell (MSC) origin that are driven by EWS/ETS fusions, most commonly EWS/FLI1. EWS/FLI1 initiates sarcomagenesis by hijacking normal MSC differentiation. Importantly, menin plays an essential role in early mesenchymal development, where it contributes to both lineage commitment and osteoblastic differentiation. We have shown that menin is over-expressed by Ewing sarcoma relative to MSC and that loss of menin results in loss of tumorigenicity. However, the mechanisms by which menin exerts its oncogenic effects in these tumor cells remain unknown. Our preliminary data identified the serine synthesis pathway (SSP) as a key downstream target of menin in Ewing sarcoma. We have confirmed that the SSP is hyper- activated in Ewing sarcoma in a menin-dependent manner, and that inhibition of PHGDH, the rate limiting enzyme in the SSP, results in profound loss of Ewing sarcoma viability, revealing a key dependence of Ewing sarcoma on the pathway. Our data also suggest that EWS/FLI1 itself contributes to activation of PHGDH and the SSP, and that transcription of a subset of EWS/FLI1-induced targets is dependent on menin. These data collectively support the hypothesis that menin functions as an oncogenic hub in Ewing sarcoma. In this proposal, we will investigate the mechanisms of menin function and test the innovative hypothesis that EWS/FLI1 promotes tumorigenesis by hijacking menin-dependent transcriptional regulation. In Aim 1 we will determine the mechanism by which menin activates the SSP and will elucidate the role of EWS/FLI1 in this process. In Aim 2 we will define the key metabolites of the SSP that contribute to tumor maintenance in order to determine why Ewing sarcoma cells are so dependent on this pathway. In Aim 3 will define genome-wide transcriptional targets of menin in MSC and Ewing sarcoma and how they are impacted by EWS/FLI1. The proposed studies will advance fundamental knowledge of the biologic underpinnings of Ewing sarcoma and discover how EWS/FLI1 hijacks normal menin physiology to promote oncogenesis. It is goal that these insights will provide opportunities to develop novel, and less toxic, menin-directed therapies for Ewing sarcoma.